Clonagem e expressão da Balternina, uma serinoprotease de Bothrops alternatus (Urutu), em sistema de células de bactéria e de levedura
Autor(a) principal: | |
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Data de Publicação: | 2019 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFU |
Texto Completo: | https://repositorio.ufu.br/handle/123456789/26317 http://dx.doi.org/10.14393/ufu.di.2019.2226 |
Resumo: | Despite their toxicity, snake venoms are composed of a series of organic and non-organic components that show activities against virus and bacteria, as well as therapeutic activities in thrombotic disorders. Thus, the characterization of biomolecules present in venoms has shown to be important to develop alternative therapies. Bhalternin, a toxin from Bothrops alternatus, has been described as a thrombin-like enzyme and has a therapeutic potential in cases of thrombotic disorders. So, the present study had as objective to perform cloning and expression of Bhalternin in bacterial and yeast systems. The mature Bhalternin coding sequence was cloned into pGS-21a e pPICZα A vectors and, after confirmation of the nucleotide sequences by Sanger Method, the plasmids that were constructed, pPGSBH2 e pPαABH9, were utilized in the transformation of Escherichia coli (strain BL21-Codon-Plus(DE3)-RIPL) and the Pichia pastoris (strain X-33), respectively. Different colonies, which showed presence of plasmid DNA by Polymerase Chain Reaction, were purified, submitted to the expression process and, then, evaluated for Bhalternin expression by protein eletrophoresis and Western blotting. In the bacterial system, a protein band corresponding to the expected molecular weight of Bhalternin expressed as fusion protein with glutathione S-transferase (GST) was observed in the supernatant and in the precipitate of bacterial lysate. However, in yeast system, it was not possible to identify a protein band corresponding to the secreted form of Bhalternin. However, in ELISA it was observed levels of expression in yeast system. Still, the coagulation test was carried out and, although a bacterial sample obtained by treating the lysate precipitate with 3 M urea showed activity, the result was inconclusive, since a sample obtained with the control clone (transformed with the vector) also showed activity. In the azocaseinolytic test, all results samples showed activity, including samples obtained with control clones. In the case of yeast expression system, the results seem to indicate low expression of Bhalternin, even though codons optimized for P. pastoris expression were used. On the other hand, in bacterial system, solubilization of Bhalternin with urea was necessary and the treatment may have caused changes in its activities. Therefore, further tests should be performed in order to obtain Bhalternin as soluble form and to evaluate its therapeutic potential in thrombotic disorders, as well as, its antiviral, antibacterial, and antiparasitic activities. |
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Clonagem e expressão da Balternina, uma serinoprotease de Bothrops alternatus (Urutu), em sistema de células de bactéria e de leveduraCloning and Expression of Balternina, a Serine protease from Bothrops alternatus (Urutu), in Bacterial and Yeast Expression SystemsPeçonhaSnake venomSerinoproteaseSerine proteaseBalternina recombinanteRecombinant BhalterninSistemas de expressãoExpression systemsCNPQ::CIENCIAS BIOLOGICASDespite their toxicity, snake venoms are composed of a series of organic and non-organic components that show activities against virus and bacteria, as well as therapeutic activities in thrombotic disorders. Thus, the characterization of biomolecules present in venoms has shown to be important to develop alternative therapies. Bhalternin, a toxin from Bothrops alternatus, has been described as a thrombin-like enzyme and has a therapeutic potential in cases of thrombotic disorders. So, the present study had as objective to perform cloning and expression of Bhalternin in bacterial and yeast systems. The mature Bhalternin coding sequence was cloned into pGS-21a e pPICZα A vectors and, after confirmation of the nucleotide sequences by Sanger Method, the plasmids that were constructed, pPGSBH2 e pPαABH9, were utilized in the transformation of Escherichia coli (strain BL21-Codon-Plus(DE3)-RIPL) and the Pichia pastoris (strain X-33), respectively. Different colonies, which showed presence of plasmid DNA by Polymerase Chain Reaction, were purified, submitted to the expression process and, then, evaluated for Bhalternin expression by protein eletrophoresis and Western blotting. In the bacterial system, a protein band corresponding to the expected molecular weight of Bhalternin expressed as fusion protein with glutathione S-transferase (GST) was observed in the supernatant and in the precipitate of bacterial lysate. However, in yeast system, it was not possible to identify a protein band corresponding to the secreted form of Bhalternin. However, in ELISA it was observed levels of expression in yeast system. Still, the coagulation test was carried out and, although a bacterial sample obtained by treating the lysate precipitate with 3 M urea showed activity, the result was inconclusive, since a sample obtained with the control clone (transformed with the vector) also showed activity. In the azocaseinolytic test, all results samples showed activity, including samples obtained with control clones. In the case of yeast expression system, the results seem to indicate low expression of Bhalternin, even though codons optimized for P. pastoris expression were used. On the other hand, in bacterial system, solubilization of Bhalternin with urea was necessary and the treatment may have caused changes in its activities. Therefore, further tests should be performed in order to obtain Bhalternin as soluble form and to evaluate its therapeutic potential in thrombotic disorders, as well as, its antiviral, antibacterial, and antiparasitic activities.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorDissertação (Mestrado)As peçonhas são compostas por uma série de componentes orgânicos e não-orgânicos que parecem mostrar atividade contra vírus e bactéria, bem como ação terapêutica em desordens trombóticas. Portanto, a caracterização de biomoléculas presentes nas peçonhas é importante para o desenvolvimento de terapias alternativas. A Balternina, uma toxina presente na peçonha de Bothrops alternatus, é uma enzima semelhante à trombina e possui um importante papel em casos de desordens trombóticas. Tendo em vista isso, o presente estudo teve como objetivo realizar a clonagem e expressão da Balternina em sistema de células de bactéria e de levedura. A sequência codificante da Balternina madura foi clonada nos vetores pGS-21a e pPICZα A e, após confirmação nucleotídica pelo método de Sanger, os plasmídeos pGSBH2 e pPαABH9 construídos foram utilizados para a transformação da Escherichia coli (cepa BL21-CodonPlus (DE3)-RIPL) e Pichia pastoris (cepa X-33), respectivamente. Diferentes colônias que apresentaram a amplificação do DNA plasmidial pela Reação em Cadeia da Polimerase (PCR), foram purificadas, submetidas ao processo de expressão e, em seguida, foram avaliadas por eletroforese de proteínas e Western blotting. No sistema bacteriano, foi observada uma banda proteica correspondente a massa molecular da Balternina expressa com a proteína de fusão Glutationa-S-Transferase (GST), tanto na fração do sobrenadante quanto no pellet. Já no sistema de levedura, não foi possível identificar uma banda proteica correspondente a Balternina. Porém, ao avaliar a expressão por Ensaio de Imunoabsorção Enzimática (ELISA), foi possível identificar níveis de expressão em sistema de levedura. Ainda, no teste de coagulação realizado, o pellet do clone bacteriano tratado com ureia 3 M mostrou atividade, mas como o clone controle tratado com ureia 8 M também demonstrou atividade, os resultados foram inviabilizados. No teste de atividade azocaseinolítica, todas as amostras demonstraram atividade, inclusive as amostras controle. No caso da expressão em levedura, os resultados sugerem uma baixa expressão da Balternina, mesmo tendo sido realizada a otimização de códons para P. pastoris. Por outro lado, no sistema bacteriano, foi requerido o tratamento com ureia e isso causou alterações na atividade da proteína. Contudo, ainda é necessário realizar mais testes, modificando condições de cultivos, bem como realizar a purificação da Balternina presente no sobrenadante dos clones bacterianos, para que assim seja possível obter melhores resultados quanto a atividade enzimática da Balternina.Universidade Federal de UberlândiaBrasilPrograma de Pós-graduação em Imunologia e Parasitologia AplicadasYokosawa, JonnyCosta, Júnia de OliveiraDias, Edigar Henrique VazPolli, Mayara Garcia2019-07-24T19:09:50Z2019-07-24T19:09:50Z2019-07-18info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfPOLLI, Mayara Garcia. Clonagem e expressão da Balternina, uma serinoprotease de Bothrops alternatus (Urutu), em sistema de células de bactéria e de levedura. 2019. 45 f. Dissertação (Mestrado em Imunologia e Parasitologia Aplicadas) - Universidade Federal de Uberlândia, Uberlândia, 2019. DOI http://dx.doi.org/10.14393/ufu.di.2019.2226.https://repositorio.ufu.br/handle/123456789/26317http://dx.doi.org/10.14393/ufu.di.2019.2226porAttribution-NonCommercial-NoDerivs 3.0 United Stateshttp://creativecommons.org/licenses/by-nc-nd/3.0/us/info:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFUinstname:Universidade Federal de Uberlândia (UFU)instacron:UFU2020-02-12T06:07:26Zoai:repositorio.ufu.br:123456789/26317Repositório InstitucionalONGhttp://repositorio.ufu.br/oai/requestdiinf@dirbi.ufu.bropendoar:2020-02-12T06:07:26Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU)false |
dc.title.none.fl_str_mv |
Clonagem e expressão da Balternina, uma serinoprotease de Bothrops alternatus (Urutu), em sistema de células de bactéria e de levedura Cloning and Expression of Balternina, a Serine protease from Bothrops alternatus (Urutu), in Bacterial and Yeast Expression Systems |
title |
Clonagem e expressão da Balternina, uma serinoprotease de Bothrops alternatus (Urutu), em sistema de células de bactéria e de levedura |
spellingShingle |
Clonagem e expressão da Balternina, uma serinoprotease de Bothrops alternatus (Urutu), em sistema de células de bactéria e de levedura Polli, Mayara Garcia Peçonha Snake venom Serinoprotease Serine protease Balternina recombinante Recombinant Bhalternin Sistemas de expressão Expression systems CNPQ::CIENCIAS BIOLOGICAS |
title_short |
Clonagem e expressão da Balternina, uma serinoprotease de Bothrops alternatus (Urutu), em sistema de células de bactéria e de levedura |
title_full |
Clonagem e expressão da Balternina, uma serinoprotease de Bothrops alternatus (Urutu), em sistema de células de bactéria e de levedura |
title_fullStr |
Clonagem e expressão da Balternina, uma serinoprotease de Bothrops alternatus (Urutu), em sistema de células de bactéria e de levedura |
title_full_unstemmed |
Clonagem e expressão da Balternina, uma serinoprotease de Bothrops alternatus (Urutu), em sistema de células de bactéria e de levedura |
title_sort |
Clonagem e expressão da Balternina, uma serinoprotease de Bothrops alternatus (Urutu), em sistema de células de bactéria e de levedura |
author |
Polli, Mayara Garcia |
author_facet |
Polli, Mayara Garcia |
author_role |
author |
dc.contributor.none.fl_str_mv |
Yokosawa, Jonny Costa, Júnia de Oliveira Dias, Edigar Henrique Vaz |
dc.contributor.author.fl_str_mv |
Polli, Mayara Garcia |
dc.subject.por.fl_str_mv |
Peçonha Snake venom Serinoprotease Serine protease Balternina recombinante Recombinant Bhalternin Sistemas de expressão Expression systems CNPQ::CIENCIAS BIOLOGICAS |
topic |
Peçonha Snake venom Serinoprotease Serine protease Balternina recombinante Recombinant Bhalternin Sistemas de expressão Expression systems CNPQ::CIENCIAS BIOLOGICAS |
description |
Despite their toxicity, snake venoms are composed of a series of organic and non-organic components that show activities against virus and bacteria, as well as therapeutic activities in thrombotic disorders. Thus, the characterization of biomolecules present in venoms has shown to be important to develop alternative therapies. Bhalternin, a toxin from Bothrops alternatus, has been described as a thrombin-like enzyme and has a therapeutic potential in cases of thrombotic disorders. So, the present study had as objective to perform cloning and expression of Bhalternin in bacterial and yeast systems. The mature Bhalternin coding sequence was cloned into pGS-21a e pPICZα A vectors and, after confirmation of the nucleotide sequences by Sanger Method, the plasmids that were constructed, pPGSBH2 e pPαABH9, were utilized in the transformation of Escherichia coli (strain BL21-Codon-Plus(DE3)-RIPL) and the Pichia pastoris (strain X-33), respectively. Different colonies, which showed presence of plasmid DNA by Polymerase Chain Reaction, were purified, submitted to the expression process and, then, evaluated for Bhalternin expression by protein eletrophoresis and Western blotting. In the bacterial system, a protein band corresponding to the expected molecular weight of Bhalternin expressed as fusion protein with glutathione S-transferase (GST) was observed in the supernatant and in the precipitate of bacterial lysate. However, in yeast system, it was not possible to identify a protein band corresponding to the secreted form of Bhalternin. However, in ELISA it was observed levels of expression in yeast system. Still, the coagulation test was carried out and, although a bacterial sample obtained by treating the lysate precipitate with 3 M urea showed activity, the result was inconclusive, since a sample obtained with the control clone (transformed with the vector) also showed activity. In the azocaseinolytic test, all results samples showed activity, including samples obtained with control clones. In the case of yeast expression system, the results seem to indicate low expression of Bhalternin, even though codons optimized for P. pastoris expression were used. On the other hand, in bacterial system, solubilization of Bhalternin with urea was necessary and the treatment may have caused changes in its activities. Therefore, further tests should be performed in order to obtain Bhalternin as soluble form and to evaluate its therapeutic potential in thrombotic disorders, as well as, its antiviral, antibacterial, and antiparasitic activities. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019-07-24T19:09:50Z 2019-07-24T19:09:50Z 2019-07-18 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
POLLI, Mayara Garcia. Clonagem e expressão da Balternina, uma serinoprotease de Bothrops alternatus (Urutu), em sistema de células de bactéria e de levedura. 2019. 45 f. Dissertação (Mestrado em Imunologia e Parasitologia Aplicadas) - Universidade Federal de Uberlândia, Uberlândia, 2019. DOI http://dx.doi.org/10.14393/ufu.di.2019.2226. https://repositorio.ufu.br/handle/123456789/26317 http://dx.doi.org/10.14393/ufu.di.2019.2226 |
identifier_str_mv |
POLLI, Mayara Garcia. Clonagem e expressão da Balternina, uma serinoprotease de Bothrops alternatus (Urutu), em sistema de células de bactéria e de levedura. 2019. 45 f. Dissertação (Mestrado em Imunologia e Parasitologia Aplicadas) - Universidade Federal de Uberlândia, Uberlândia, 2019. DOI http://dx.doi.org/10.14393/ufu.di.2019.2226. |
url |
https://repositorio.ufu.br/handle/123456789/26317 http://dx.doi.org/10.14393/ufu.di.2019.2226 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
Attribution-NonCommercial-NoDerivs 3.0 United States http://creativecommons.org/licenses/by-nc-nd/3.0/us/ info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Attribution-NonCommercial-NoDerivs 3.0 United States http://creativecommons.org/licenses/by-nc-nd/3.0/us/ |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Uberlândia Brasil Programa de Pós-graduação em Imunologia e Parasitologia Aplicadas |
publisher.none.fl_str_mv |
Universidade Federal de Uberlândia Brasil Programa de Pós-graduação em Imunologia e Parasitologia Aplicadas |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UFU instname:Universidade Federal de Uberlândia (UFU) instacron:UFU |
instname_str |
Universidade Federal de Uberlândia (UFU) |
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UFU |
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UFU |
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Repositório Institucional da UFU |
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Repositório Institucional da UFU |
repository.name.fl_str_mv |
Repositório Institucional da UFU - Universidade Federal de Uberlândia (UFU) |
repository.mail.fl_str_mv |
diinf@dirbi.ufu.br |
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1805569631108202496 |